1. Field of the Invention
This invention relates to improvements in methods and apparatus for measuring the hemoglobin content of individual red blood cells of a whole blood sample. More particularly, this invention relates to a method for measuring a red blood cell's hemoglobin content on the basis of its reflectivity to radiation at a particular wavelength or wavelengths.
2. The Prior Art
In diagnosing various types of anemias and other blood disorders, as well as in the monitoring of medical treatments, it is necessary to evaluate certain properties of an individual's red blood cells. Those properties of red cells that are routinely reported include: the number of red cells per unit volume of blood (i.e., the red blood cell count, or RBC), the volume percentage of red cells in a whole blood sample (referred to as the Packed Cell Volume, or PCV), the amount of hemoglobin per unit volume of whole blood (referred to as the Hemoglobin Concentration, or [Hgb]), the average size of the red cells (Mean Cell Volume, or MCV), the distribution of the red cell sizes (Red Cell Distribution Width, or RDW), the average amount of hemoglobin in each blood cell (Mean Cell Hemoglobin or, MCH), and the average concentration of hemoglobin within the red blood cells as a whole (Mean Cell Hemoglobin Concentration, or MCHC). Of these particular parameters, most hematology analyzers directly measure only three: RBC, [Hgb], and MCV. The other parameters are calculated from the directly measured parameters.
In addition to the above-noted parameters, other red cell parameters are also useful in fully assessing a blood sample to provide an early diagnosis and/or treatment of disease. Red cells of the same blood sample can substantially differ in their hemoglobin content. When a smear of red cells is viewed under a microscope, the amount of hemoglobin in each red cell correlates well with its color; the brighter red in color, the more hemoglobin within the cell. Having knowledge of the statistical distribution of the individual cell hemoglobin concentrations within a population adds significant information concerning the health of a patient. Red cells with decreased hemoglobin concentration are called hypochromic, while red cells with increased hemoglobin concentration are termed hyperchromic. Populations with an increased distribution of hemoglobin concentrations (i.e., a wide disparity of concentrations) are classified as polychromatophilic. Hyperchromic red cells have altered flow properties and have been suggested as the cellular cause of diseases such as sickle cell anemia.
To provide the statistical distribution information noted above, as well as to provide a more accurate determination of the MCH and MCHC parameters, it is known to measure the hemoglobin concentration of a whole blood sample on a cell-by-cell basis. Such measurements are commonly effected by flow cytometric techniques in which the forward light-scattering (FLS) properties, DC volume (V) and/or RF conductivity (C) of individual cells are determined as each cell passes, one-by-one, through a tiny sensing aperture formed in a cytometric flow cell. See, for example, the respective disclosures of U.S. Pat. Nos. 4,735,504 and 5,194,909, both issued to D. H. Tycko, and the commonly assigned U.S. Pat. No. 5,194,909 issued to R. S. Frank et al.
In the '504 patent to Tycko, “sphered” red cells (i.e., red cells that have been treated to render them substantially spherical in shape) are illuminated by a laser beam as they pass single file through the sensing aperture of an optical (transparent) flow cell. The level of forwardly scattered light from each cell is monitored within two angular regions, and an appropriate algorithm is used to determine each cell's hemoglobin concentration and volume on the basis the two light scatter measurements made. In an article entitled “Flow-Cytometric Light Scattering Measurement of Red Blood Cell Volume and Hemoglobin Concentration” by D. H. Tycko, Applied Optics, Vol. 24, No. 9 (1985), it is noted that the optical measurements relating to volume and HC are extremely complex and interrelated, and require a relatively complex and sophisticated optical system to implement. Additionally, this method requires an absolute calibration of the two optical channels which cannot be done by commonly available latex microspheres. Also, it is noted that the light scatter intensities are non-linear functions of both V and HC, and require considerable computing resources.
In the '909 of Tycko, the patentee improves upon the technique described above by measuring the volume of the sphered red blood cells independently from the optical measurement, thereby reducing the optical complexity and computational requirements. Volume is measured by the standard Coulter Principle according to which a change in a low frequency or DC current (caused to pass through the sensing aperture of a flow cell simultaneously with the passage of cells) indicates the size (volume) of the cell passing through the aperture. In both the '504 and '909 patents, the patentee relies on the supposition that the index of refraction of individual cells, which determines the forward light scattering characteristics, is directly related to the hemoglobin concentration of the cells. This supposition, however, is not necessarily true. It is known that at least about 95% of the interior of a red blood cell is a mixture of hemoglobin molecules and water molecules, and the relative proportions of these two molecules is variable; thus the index or refraction, is variable. Likewise, the remaining 5% of the interior volume of the red blood cell is composed mainly of salts which have a large effect on the index or refraction, especially, when the hemoglobin concentration is very low. Never-the-less, Tycko teaches that by determining the index of refraction by measuring the forward light-scatter intensity within a predetermined angular range, and using the volume of the cell as determined by the DC current measurement through the sensing aperture, the hemoglobin concentration of the cell can be computed.
In the '309 patent of Frank and Wyatt, the hemoglobin concentration is determined by a non-optical technique in which changes in DC and RF currents passing through the sensing aperture simultaneously with the passage of the individual red cells are monitored. While the hemoglobin concentration correlates well with the cell's conductivity, the flow cell aperture must be relatively small in cross-section (preferably about 50×50 microns) in order to achieve a readily detectable change in the RF current. This requirement impacts the overall reliability of the device, since the small aperture can be subject to frequent blockage caused by clumps of cells or debris in the system. Another disadvantage of this technique is the requirement that the conductivity of the diluting fluid required in making the measurement be well controlled to achieve reproducible results.